Hypoxia and inflammation often occur concomitantly, and together, result in profound changes in tissue metabolism. Such "inflammatory hypoxia" results, at least in part, from active recruitment of energy and oxygen demanding inflammatory cell types. In the past several years, we have focused on defining molecular pathways and functional targets for endothelial hypoxia. Results from these studies have identified a novel series of pathways in which hypoxia (both in vitro and in vivo) drives the coordinated metabolism of extracellular nucleotides. These studies have identified endogenous anti-inflammatory properties of hypoxia-induced adenosine (Ado) signaling. Initial insight gained from global transcriptional arrays identified the prominent regulation of surface enzymes (CD39, CD73, adenosine deaminase), nucleotide transporters (ENT1 and ENT2) and Ado receptors (esp. A2B subtype) critical for the regulated metabolism and signaling of Ado. Central to this pathway was the identification of hypoxia-inducible factor (HIF) binding and regulation of each of these gene promoters, thus identifying HIF as an endogenous anti-inflammatory molecule. Ongoing studies have revealed that Ado functions as an endogenous protective agent mediating multiple anti-inflammatory endpoints. Ongoing studies have identified the basis of Ado-mediated anti-inflammation. Studies addressing regulation of HIF-11 and NF-:B revealed that Ado activates HIF-11 and inhibits NF-:B through the active deneddylation of Cullins, a family of proteins critical for the recruitment ubiquitin ligases. Based on these preliminary studies, we hypothesize that Ado functions as an endogenous anti-inflammatory through direct actions on vascular HIF and NF-:B. Three specific aims are directed at testing this hypothesis: In Specific Aim 1, we will probe the contribution of individual Ado receptor subtypes to Ado actions on HIF and NF-:B via Cullin deneddylation. Specific Aim 2 will extend extensive preliminary data to elucidate mechanisms of Ado-mediated Cullin deneddylation. Specific Aim 3 will build on preliminary studies to define endpoints of Ado-mediated Cullin deneddylation via PHD inhibition. The overall aim of this proposal is to elucidate the metabolic signaling events mediating Ado vascular responses during inflammatory hypoxia. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Studies outlined here build on existing work related to acute and chronic inflammatory disease. In particular, these studies are designed to identify, test and unify hypotheses that adenosine functions as an endogenous anti-inflammatory through distinct mechanisms.